DNA Replication/ PCR/ Restriction Digests

Objectives of this Lab Exercise (over the next couple

of weeks):

Polymerase Chain Reaction (PCR)

Restriction Enzyme Digestion

Gel Electrophoresis

Today: Extract the DNA and set up the PCR reactions

DNA Replication

Identify your specific genotype for the “PTC” tasting

gene

The Polymerase Chain Reaction (PCR)

Taq polymerase

Thermus aquaticus

Producing identical copies of

a DNA segment

(typically smaller than a gene)

DNA amplification

Thermocycler

Ingredients

Steps

The Structure of DNA

1) How is the genetic information copied?

2) How does DNA store the genetic

information?

3) How is the genetic information passed

from generation to generation?

The Structure of a Single DNA Strand

o Sugar-phosphate backbone

o Monomers: Nucleotides

o Nitrogenous bases:

purines & pyrimidines

o 5’ and 3’ ends

The Double Helix Base-Pairing

Uniform Width of the DNA Double Helix

The Double Helix

Base-Pairing/

Provided a basis for copying DNA but

the mechanism was not understood

What type of

bonds connects

two nucleotides

together?

Three Alternative Hypothesis

Conservative Replication

Semi-Conservative Replication

Dispersive Replication

Meselson and Stahl’s Experiment

Density Gradient Centrifugation

100% (saturated) NaCl

50% saturated NaCl

25% saturated NaCl

dH2O

Meselson and Stahl’s Experiment

Density Gradient Centrifugation

Red: 15N

Blue: 14N

Manipulation of DNA

Molecular Biology Techniques

Restriction enzymes: endonucleases that recognize a unique nucleotide sequence and make

cuts in the sugar-phosphate backbone of each DNA strand

Single Nucleotide Polymorphisms

(SNPs)

One nucleotide difference in a DNA sequence among individuals

G G C G G G C A C T

G G C G G C C A C T

Can be used for genotyping

DNA Replication

Overview

Origins of Replication - Eukaryotes

May have hundreds or even thousands of replication origins

Replication begins at specific sites where parent strands separate to form replication

bubbles

Replication bubbles expand laterally as DNA replication proceeds

Replication bubbles fuse and synthesis of the daughter strands is complete

sister chromatids!!!

How about

prokaryotes?

The Cast of Characters

Elongation of new DNA at a replication fork:

DNA polymerases add nucleotides to the 3 end of a growing strand

The Role of DNA Polymerase

How does the antiparallel structure of the double helix affect

replication?

DNA polymerases add nucleotides only to the free 3 end of a growing strand

Along one template strand of DNA, the leading strand,

DNA polymerase III can synthesize a complementary strand

continuously, moving toward the replication fork

To elongate the other new strand of DNA, the lagging strand

DNA polymerase III must work in the direction away from the

replication fork

The lagging strand is synthesized as a series of segments called

Okazaki fragments, which are joined together by DNA ligase

Priming Replication

DNA Polymerases cannot initiate replication!

They can only add nucleotides to the 3’end of a growing strand

Need to have a ‘primer’ in place

RNA primer

Only one primer is needed for replication of the leading strand!

For the lagging strand, each Okazaki fragment must have its own primer

SSBPsHelicase

Topoisomerase

The leading strands are faded out

to help you focus on synthesis

of the lagging strand

SSBPs HelicaseTopoisomerase

The leading strands are faded out

to help you focus on synthesis

of the lagging strand

1. Primer added. Primase synthesizes RNA primer.

Primase

RNA

primer

2. First fragment synthesized. DNA polymerase III works in 5 → 3 direction,synthesizing first Okazaki fragment of lagging strand.

DNA polymerase III

Sliding clamp

Okazaki fragment

3. Second fragment synthesized. Primase and DNA polymerase IIIsynthesize another Okazaki fragment.

1st Okazaki fragment

2nd Okazaki fragment

4. Primer replaced. DNA polymerase I removes ribonucleotides of primer,replaces them with deoxyribonucleotides in 5 → 3 direction.

DNA polymerase I

5. Gap closed. DNA ligase closes gap in sugar-phosphate backbone.

DNA ligase

Telomeres Shorten during Normal DNA Replication

1. Helicase unwinds end of DNA helix (at end of chromosome).

5

5

3

3

CHROMOSOME SHORTENING DURING NORMAL DNA REPLICATION

RNA primer

33 5

SSBPs Helicase

Leading strand

Lagging strand

Sliding clampDNA polymerase

5

5

End of chromosome

2. DNA polymerase completes the leading strand. Primase synthesizes RNA primer at endof lagging strand.

Telomerase Prevents Shortening of Telomeres

5

DNA polymerase

TELOMERE REPLICATION

Missing DNA on

lagging strand

Sliding clamp

55

RNA primer

3

3

53

3Telomerase with itsown RNA template

5

5

5

3

3

1. When the RNA primer is removed from the5 end of the lagging strand (see Figure 14.14),a strand of parent DNA remains unreplicated.

4. Primase, DNA polymerase, and ligase thensynthesize the lagging strand in the 5→3direction, restoring the original length of thechromosome.

2. Telomerase binds to the “overhanging” sectionof single-stranded DNA. Telomerase addsdeoxyribonucleotides to the end of the parentDNA, extending it.

3. Telomerase moves down the DNA strand andadds additional repeats.

The Cast of Characters

ampR

• aproximately 5,400 bp

Bacterial Transformation

GFP are used to monitor the activity of altered genes

Transformation

experiment

Gene CloningCLONING -- Producing identical copies of a DNA segment

(gene)

Figure 20.2